Tesfaye Desalegn

Anticonvulsant activity of Soxhlet leaf extracts of Ajuga integrifolia
Buch.Ham ex D.Don (Lamiaceae) in mice
By: Tesfaye Desalegn (B.Pharm)
A thesis submitted to the Department of Pharmacology and Clinical

Pharmacy, School of Pharmacy, College of Health Sciences, Addis
Ababa University in partial fulfillment of the requirements for Master
of Science degree in Pharmacology
November, 2021
Addis Ababa University
Addis Ababa, Ethiopia

ADDIS ABABA UNIVERSITY
SCHOOL OF GRADUATE STUDIES
This is to certify that the thesis prepared by Tesfaye Desalegn entitled “Anti-convulsant
activity of Soxhlet leaf extracts of Ajuga integrifolia Buch.Ham ex D.Don (Lamiaceae) in
mice” and submitted in partial fulfillment for the requirements of Master of Science degree in
Pharmacology complies with the regulation of the university and meets the respected
standards with respect to originality and quality
Signed by examining committee
Name signature date
Examiner (internal) _____________________ __________ ___________
Examiner (external) ______________________ ___________ __________
Adviser Prof. Ephrem Engidawork __________
_______________________________________________________
Chair, Department of program coordinator

ABSTRACT
Epilepsy is one of the most common chronic neurological conditions that affect 70 million
people in different parts of the world. The leaves of Ajuga integrifolia Buch.ham ex D Don
have been used as anti-convulsant remedy in Ethiopian traditional medicine. However, the
evidence supporting this claim is sparse in the literature. This study was conducted to add to
the existing body of knowledge about the anti-convulsant activity of the plant. To this effect
successive Soxhlet extraction was performed using n-hexane, ethyl acetate, methanol and
water.
Anti-convulsant activity of the extracts was investigated in both acute (pentylenetetrazole,

PTZ; and maximal electroshock, MES) and chronic (PTZ kindling) seizure models. For the
acute model, various doses of the extract (100mg/kg, 200mg/kg and 400mg/kg) were
administered. Positive controls received sodium valproate (200mg/kg) for PTZ and
phenytoin (25mg/kg) for MES. Distilled water or 2% tween 80 was used for negative
controls. Kindling was induced by repeated alternate day intra-peritoneal administration of
sub-convulsive dose of PTZ (35 mg/kg) for 13 days and the most active extract (ethyl
acetate) was tested in this model. Parameters including onset of clonus and duration of hind
limb tonic extension were recorded. Moreover, total alkaloid, flavonoid and phenol contents
of the most active extract were determined.
Treatment of mice with ethyl acetate extract produced a superior effect among all solvent
extracts in both PTZ and MES model. The mean latency to clonic seizure was significantly
increased (p<0.01) with all doses of ethyl acetate extract in PTZ test compared to control and
n.hexane extract ranked next to ethyl acetate extract in increasing onset of clonus. It
significantly increased mean onset of clonus compared to controls, with a maximum increase
(12.67min, p<0.001) displayed by HA400 mg/kg. Methanol extract at 200mg/kg and 400
mg/kg also significantly delayed onset of clonus (p<0.001) in PTZ model.Once again, all
doses of ethyl acetate extract of the study plant significantly reduced (p<0.001) the mean
duration of hind limb tonic extension in MES test compared to control. Hexane extract at 200
mg/kg and 400 mg/kg also significantly reduced (p<0.001) duration of hind limb tonic
extension. Methanol extract at 200mg/kg and 400 mg/kg also significantly reduced (p<0.01)
I
mean duration of hind limb tonic extension (HLTE) compared to control in MES test.
Aqueous extract at all doses was devoid of any anti-convulsant effect in both models. A
similar type of study done on the leaf crude extract and solvent fractions collected from
different geographical location also reported anti-convulsant activity of the plant in acute
seizure models. Treatment of mice with 200mg/kg and 400mg/kg of ethyl acetate extract
along with alternate day PTZ injection significantly protected(p<0.01 for 200mg/kg and
p<0.001 for 400 mg/kg) against PTZ induced kindling compared to controls in chronic
model. Ethyl acetate extract of the plant was found to contain 10.002±0.119 mg atropine
equivalent per gram of dry extract of alkaloids, 9.045 ±0.8445 mg quercetin equivalent /g of
dry weight extract of flavonoids and 21.928 ±1.118 mg gallic acid equivalent / g of dry
weight of extract of phenols. This study indicated that the plant has anti-convulsant activity
in both acute and chronic model and it could be a potential source to develop a new anti-
epileptic drug for pharmacoresistant epilepsy.
Key words: Ajuga integrifolia, Anti-convulsant, Epilepsy, kindling, phytoconstituents,

Seizure
II
ACKNOWLEDGEMENTS
First of all, I would like to thank the almighty God who has given me the strength while
writing this thesis.
I would also like to thank my adviser, Prof. Ephrem Engidawork, who provided me with
encouragement. His Scientific insight and approach nurtured my ability to effectively frame
Scientific problems and design experiments. These educational experiences helped me
realize which question to ask and determine not only the solutions but also the subsequent
questions spawned from these answers.
I am also so grateful to Dr. Samson Sahilu, Mrs. Fantu Assefa, Mr.Yohannes Tsegiye and
Mrs. Etetu Mamo for their cooperation in providing materials and chemicals for the
experimental study. My since gratitude also goes to Mr. Besufikad Abebe who helped me a
lot during the extraction process by providing and setting the Soxhlet apparatus used for
extraction and quantification of secondary metabolites. I would also like to thank Mr.
Sintayehu Getachew for his support during plant material collection and my classmate Hana
Saif for her invaluable support during the entire study.
I wholeheartedly like to thank my wife, family and friends, especially Dr. Biniyam Dawit
and Mr.Tarekegn Motuma for their support, encouragement and care during my stay at Addis
Ababa University. Finally, I express my heartfelt thanks to Addis Ababa University and
Madda Walabu University for granting me the financial support and all the necessary
material for the entire study.
III
Table of Contents
ABSTRACT ............................................................................................................................................ I
ACKNOWLEDGEMENTS .................................................................................................................. III
LIST OF ABBREVIATIONS AND ACRONYMS .............................................................................. VI
LIST OF TABLES ............................................................................................................................... VII
LIST OF FIGURES ............................................................................................................................. VIII
1. INTRODUCTION .............................................................................................................................. 1
1.1. Overview of Epilepsy .................................................................................................................. 1
1.2. Epidemiology of Epilepsy ........................................................................................................... 2
1.3. Pathophysiology of Epilepsy ....................................................................................................... 2
1.4. Management of Epilepsy ............................................................................................................. 6
1.4.1. Non-pharmacological treatment ........................................................................................... 6
1.4.2. Pharmacological treatment ................................................................................................... 7
1.4.3. Herbal treatment ................................................................................................................... 8
1.5. Animal models of epilepsy .......................................................................................................... 9
1.6. Overview of the experimental plant .......................................................................................... 12
1.7. Rationale of the study ................................................................................................................ 16
2. OBJECTIVES .................................................................................................................................. 18
2.1. General objective ....................................................................................................................... 18
2.2. Specific objectives ..................................................................................................................... 18
3. EXPERIMENTAL METHODS ....................................................................................................... 19
3.1. Drugs and chemicals.................................................................................................................. 19
3.2. Experimental Animals ............................................................................................................... 19
3.3. Collection of plant material ....................................................................................................... 19
3.4. Preparation of the extracts ......................................................................................................... 20
3.5. Grouping and dosing of animals................................................................................................ 20
IV
3.6. Anti-convulsant activity test ...................................................................................................... 21
3.6.1. PTZ induced seizure ........................................................................................................... 21
3.6.2. MES induced seizure .......................................................................................................... 22
3.6.3. PTZ induced Kindling ........................................................................................................ 22
3.7. Quantification of phytochemical constituents ........................................................................... 23
3.7.1. Quantification of total flavonoids content .......................................................................... 23
3.7.2. Quantification of total phenolic content ............................................................................. 23
3.6.3. Quantification of total alkaloids ......................................................................................... 24
3.8. Data Analysis ............................................................................................................................ 24
4. RESULTS ......................................................................................................................................... 25
4.1. Anti-convulsant activity in PTZ induced seizure ...................................................................... 25
4.2. Anti-convulsant activity in MES induced seizure ..................................................................... 27
4.3. Anti-convulsant activity in PTZ kindling Model ...................................................................... 29
4.4. Quantification of Secondary Metabolites .................................................................................. 30
4.4.1. Determination of Total flavonoids content ......................................................................... 30
4.4.2. Determination of total phenolic content ............................................................................. 31
4.4. 3. Quantification of Total alkaloid content ............................................................................ 32
5. DISCUSSION .................................................................................................................................. 34
6. LIMITATION OF THE STUDY ..................................................................................................... 39
7. CONCLUSION ................................................................................................................................ 40
8. SUGGESTION FOR FUTURE WORK .......................................................................................... 41
9. REFERENCES ................................................................................................................................. 42
V
LIST OF ABBREVIATIONS AND ACRONYMS
AED Anti-epileptic Drugs

AMPA α-Amino-3-hydroxy-5-Methyl-isoxazolopropionic Acid
ANOVA Analysis of Variance
CA Cornu Ammonis
CNS Central Nervous System
DALY Disability Adjusted Life Year
DPPH 1,1-diphenyl-2-picrylhydrazyl
EEG Electroencephalography
GABA Gamma Amino Butyric Acid
ICAM Intercellular Adhesion Molecule
IL Interleukin
ILAE International League Against Epilepsy
IP Intra-peritoneal
HLTE Hind Limb Tonic Extension
MES Maximal Electro Shock
mGluR Metabotropic Glutamate Receptor
NMDA N-Methyl-D-Aspartate
MRI Magnetic Resonance Imaging
PTZ Pentylenetetrazol
SPSS Statistical Package for Social Sciences
TAC Total Alkaloid content
TFC Total Flavonoid Content
TPC Total Phenolic Content
TLE Temporal Lobe Epilepsy
Vascular
VCAM Cell Vascular Cell Adhesion Molecule
VEGF Vascular Endothelial Growth Factor
VGCCs Voltage Gated Calcium Channels
VGSC Voltage Gated Sodium Channel
VI
LIST OF TABLES
Table 1. Anti-convulsant activity of the Soxhlet leaf extracts of Ajuga integrifolia in
pentylenetetrazole induced seizure ......................................................................................... 26
Table 2. Anti-convulsant activity of the Soxhlet leaf extracts of Ajuga integrifolia in MES
induced seizure........................................................................................................................ 28
VII
LIST OF FIGURES
Figure 1: Photograph of Ajuga integrifolia Buch.Ham ex D.Don (Photo Credit: Tesfaye
Desalegn) ................................................................................................................................ 15
Figure 2: Clonic seizure exhibited after 80mg/kg subcutaneous injection of PTZ................. 21
Figure 3: Effect of Ethyl acetate extract on Pentylenetetrazole induced kindling in mice.
Animals were treated with 100mg/kg, 200mg/kg and 400mg/kg ethyl acetate extract and
200mg/kg sodium valproate along with PTZ 35mg/kg i.p every other day for 13 days. ....... 30
Figure 4: Standard curve constructed for quercetin based on absorbance measured at 415 nm.
................................................................................................................................................. 31
Figure5: Standard curve constructed for gallic acid based on absorbance measured at 765 nm.
................................................................................................................................................. 32
Figure 6: Standard curve constructed for atropine based on absorbance measured at 470 nm.
................................................................................................................................................. 33
VIII
1. INTRODUCTION
1.1. Overview of Epilepsy

Epilepsy is one of the most common chronic neurological disorder that affects many people
in different parts of the world(Thijs et al., 2019). Despite their difference, the term epilepsy
and seizure are often confusing(Scharfman, 2007). As defined by the International League
Against Epilepsy( ILAE), epilepsy is a disease of the brain characterized by any of the
following conditions: (i) At least two unprovoked (or reflex) seizures occurring >24 h apart;
(ii) one unprovoked (or reflex) seizure and a probability of further seizures similar to the
general recurrence risk (at least 60%) after two unprovoked seizures, occurring over the next
10 years; (iii) presence of an epilepsy syndrome even if the risk of subsequent seizure is very
low (Fisher et al., 2014). Seizures are characterized by disturbed cerebral function caused by
abnormal, excessive, and synchronous electrical discharges in groups of cortical neurons that
may produce sub-clinical or various clinical phenomena(Fisher et al., 2017, Mosewich and
So, 1996).
Epilepsy is divided into 4 categories based on etiology: idiopathic, symptomatic, provoked,

and cryptogenic. Idiopathic epilepsy is epilepsy which is predominantly genetic or presumed
genetic origin in which there is no gross neuro-anatomic or neuro-pathologic abnormality
(epilepsy with no underlying structural brain lesion or other neurologic signs or symptoms).
Symptomatic epilepsy is epilepsy of acquired or genetic cause associated with gross
anatomic or pathologic abnormalities. It is the result of one or more identifiable structural
lesions of the brain. On the other hand epilepsy which is predominantly caused by specific
systemic or environmental factor in which there are no gross anatomic or neuro-pathologic
change is known to be provoked epilepsy whereas epilepsy of presumed symptomatic nature
in which the cause has not been identified is known as cryptogenic epilepsy(Engel Jr, 2001,
Shorvon, 2011).
Three types of seizure can be identified on the basis of clinical phenomena as it can be
evidenced by electro-encephalography (EEG). They are: generalized seizure, focal seizure
and seizure with unknown onset (Fisher et al., 2017).
1
Generalized seizure is a type of seizure that originates simultaneously on both sides of the
brain hemispheres and spreads rapidly via neuronal networks. There are a range of
generalized seizure types such as tonic-clonic (grandmal), absence (non motor), myoclonic,
tonic and atonic seizure. Focal seizure is a seizure originating from networks limited to one
hemisphere that may be discretely localized or more widely distributed. It can present with a
range of symptoms depending on site of abnormal electrical discharge in the brain. The third
type is seizure with unknown onset in which the physician is occasionally certain whether it
is focal or generalized. This is more common in health facilities where an access to brain
imaging techniques like magnetic resonance imaging (MRI) is limited. In addition to
identifying the seizure types, epilepsy can also be diagnosed by identification of an epilepsy
syndrome such as EEG changes, brain imaging abnormalities, and genetic analyses (Brodie
et al., 2018, Fisher et al., 2017, Richardson et al., 2015).
1.2. Epidemiology of Epilepsy

Epilepsy is one of the most common chronic neurologic disorders, affecting almost 70
million people worldwide. Despite it is a global disease, epilepsy has unequal distribution
and about 80% of the affected individual live in low- and middle- income countries. The
incidence and prevalence of epilepsy is higher in this area than the rest of the world and this
is due to some risk factors such as head trauma, perinatal injury and CNS infection which
are more common in poor regions, especially in rural areas(Espinosa-Jovel et al., 2018).
Studies in developing countries have reported a prevalence rate ranging from 3.7 to 57 per
100,000 person and 80%-90% of the epileptic patients in this area do not receive treatment
at all (Carpio and Hauser, 2009).
Epilepsy is also a common neurological disorder in Ethiopia with estimated prevalence of 5–

8/1000 population(Deresse and Shaweno, 2016). Another study conducted in Ethiopia
reported higher prevalence which is 29.5/1000(Almu et al., 2006).
1.3. Pathophysiology of Epilepsy

Epileptic seizure arises from an excessively synchronous electrical discharge from group of
neurons in the brain and a persistent increase of neuronal excitability is common feature of
all epileptic seizures. Although the abnormal cellular discharge can be associated with
different factors such as trauma, oxygen deprivation, tumors, infection, and metabolic
2
derangements, specific causative factors cannot be found in about half of the epileptic
patients and it is unclear which factor is responsible for the process of epileptogenesis
(Engelborghs et al., 2000). However, different experimental studies have provided insights
about postulated mechanisms responsible for the process of epileptogenesis (M Manchishi,
2018)
1.3.1. Neurotransmitters
Alteration in activity of different neurotransmitters in the brain plays a key role in the
pathogenesis of epilepsy among which gamma amino butyric acid (GABA) and glutamate
are the most common(Werner and Coveñas, 2017).
GABA is the major inhibitory neurotransmitter in the CNS and its inhibition results in
epilepsy as evidenced by different experimental epilepsy models(Khazipov, 2016). GABA is
formed within GABA ergic axon terminals, where it is discharged into neuronal connections
and acts on its receptors, GABAA and GABAB located at synaptic membranes. GABAA,
controls chloride entry into the cell, and GABAB, increases potassium conductance,
decreases calcium entry, and inhibits the presynaptic release of other transmitters.
Experimental and clinical studies indicated that GABA has a role in the pathogenesis and
treatment of epilepsy because: (i) inhibition of GABAergic function is observed in animal
models of epilepsy; (ii) GABA agonists suppress seizure and antagonists induce seizure; (iii)
benzodiazepines and barbiturates employed in the treatment of epilepsy are found to enhance
GABAergic function; and (iv) drugs that enhance synaptic activity like vigabatrin and
tiagabin are potent anti-convulsants (Treiman, 2001).
Glutamate is the predominant excitatory neurotransmitter in the brain and applies its
excitatory impact via fast ionotropic, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA),N-methyl-D-aspartate (NMDA),and kainate as well as slow metabotropic glutamate
receptors (mGluR). NMDA receptors are more commonly involved in the process of
epileptogenesis as proved by the role of NMDA receptor antagonists such as ketamine to
suppress seizure in animal models. Currently, of all antiepileptic drugs (AEDs) approved
clinically, only felbamate antagonizes NMDA receptors(Barker-Haliski and White, 2015,
Huff et al., 2016).
3
At rest NMDA receptor is blocked by Mg+2 and it is immediately removed by post-synaptic
depolarization following glutamate release rendering it permeable to both Na+ and Ca++ and
this is important for neurotransmission. The balance of glutamate and GABA is also crucial
for the normal functioning of neuronal cells. If excess glutamate is released as in post
traumatic injury, the cell membrane remains depolarized leading to increased Ca++ entry into
post –synaptic cell causing excess neuronal excitability that can lead to seizure (Guerriero et
al., 2015). In addition, changes in metabotropic glutamate receptor activity can also play an
important role in causing epilepsy(Engelborghs et al., 2000).
1.3.2. Voltage gated ion channels
Voltage gated ion channels (VGICs) such as sodium, potassium and calcium play a vital part
in the process of epileptogenesis as proven by various currently available AEDs targeting
these ion channels. Under normal physiology, these ion channels perform various activities
controlling normal action potential generation or neuronal excitability. Mutation in these ion
channels results in functional change causing hyper-excitability of neuronal cells leading to
epileptic seizure(Sancheti and Sathaye, 2013).
Voltage gated sodium channel (VGSC) is a core in the pathogenesis of epilepsy and
numerous of the commonly used AEDs like phenytoin, carbamazepine and lamotrigine block
this ion channel. Diverse experimental studies demonstrated that abnormal expression or
function of VGSCs has a part in the epileptogenesis of both inherited and acquired epilepsy.
The presence of several hundred mutations of VGSC gene that lead to inherited epileptic
syndrome is the most convincing proof indicating the ion channel plays an important role in
the pathogenesis of epilepsy(Köhling, 2002, Mantegazza et al., 2010).
Voltage gated calcium channels (VGCCs) are critical for normal physiology controlling
neurotransmitter release, cell excitability and gene expression. The VGCCs can be classified
as P/Q, N, L, R (collectively termed high voltage activated (HVA)) and T (collectively
termed low voltage activated (LVA)) based on their physical and pharmacological properties.
The L-type calcium channel, which produces long-lasting inward Ca+2 current, has five
polypeptide subunit and calcium antagonists block the 1 subunit. This type of channel is
4
documented in the area of CNS such as cortex, hippocampus, cerebellum and spinal cord.
The N (high-threshold inactivating) and T (low) type calcium channel have also been
documented in neurons. Rhythmic firing of neurons is contributed by T-type current where as
N or L-type currents are involved in the release of neurotransmitters(Ku³ak et al., 2004).
Like VGSC, VGCCs also play a pivotal role in the process of epileptogenesis as indicated by
different experimental models like: (i) kindling animal seizure model in which sub-
convulsive electrical stimulation is applied to amygdala, ultimately generates intense limbic
and clonic motor seizures; (ii) mutations of genes encoding VGCCs are associated with
tonic-clonic and behavioral hall marks of absence seizure; (iii)ablation of VGCCs are also
associated with hall marks of epilepsy; and (iv) the availability of AEDs targeted against
VDCCs like ethosuximide, which is effective against absence but not partial or generalized
tonic-clonic seizure by specifically blocking T-type VGCC(Jones, 2002, Weiss and Zamponi,
2019).
1.3.3. Changes in neuronal networks
Test model of chronic temporal lobe epilepsy (TLE) induced by the chemo-convulsant
pilocarpine as well as finding from post-surgical or postmortem human temporal lobe
specimens from patients with TLE revealed that there is a disturbance in the glial-neuronal
networks which might have a role in epileptogenesis. For example investigations of
specimen from patients with TLE revealed that there is an alteration in astrocytes
activity(Brennan et al., 2021, Coulter and Steinhäuser, 2015). Dysfunction in the
hippocampal neurons could also occur after acute seizure as proved by degeneration of
neurons in the hilus of dentate gyrus and cornu ammonis (CA1-CA3) pyramidal neurons and
loss of inhibitory GABAergic interneurons(Yin et al., 2013).
1.3.4. Inflammatory Mediators
Investigations using experimental models identified that inflammatory processes have a

crucial role in the pathogenesis of epilepsy. In rodent models of epilepsy, pharmacological or
electrical stimulation of seizures triggers the release of pro-inflammatory cytokines such as
interleukins (IL) 1β, IL-6, tumor necrosis factor (TNF) alpha, and high mobility group box 1
(HMGB1) from glial cells. In addition, in these experimental models, cytokine receptor
5
expression is upregulated in neurons as well as astrocytes and microglia cells indicating their
role in epileptogenesis. In addition to inflammatory cytokines, other inflammatory mediators
such as prostaglandins (PGs) are also markedly increased following seizure (Cerri et al.,
2017). Analysis of specimen from patients with TLE also indicated that there is up regulation
of inflammatory mediators such as vascular endothelial growth factor (VEGF),Intercellular
adhesion molecule(ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and interleukins
(ILs (5, 7,13, 22,25 and 27) (Kan et al., 2012).
1.4. Management of Epilepsy

Management of epilepsy is not an easy task and it requires a careful identification of goal of
therapy, seizure type and frequency and epilepsy syndromes. Depending on these factors one
or more of the non-pharmacological, pharmacological and herbal method of management can
be employed(Brodie and French, 2000).
1.4.1. Non-pharmacological treatment

Non-pharmacological interventions are used to manage epilepsy in conjunction with or as an
alternative to AEDs. The most commonly used interventions are diet, surgery and vagus
nerve stimulation(Jackson et al., 2015). Ketogenic diet, a restrictive high-fat, low- protein
and very low-carbohydrate has been used as an alternative to AEDS in the treatment of
pharmacoresistant epilepsy in adults. In spite of the fact that the precise mechanism of
ketogenic diets in controlling of seizure is obscure, serum ketone bodies alter neuronal
metabolic status and thought to impact the number and work of neurotransmitters through
interaction with receptors, channels and metabolic enzymes (Williams and Cervenka, 2017).
Surgical treatment to abolish seizures has been specially suggested for patients with mesial
TLE and neocortical epilepsy(Jackson et al., 2015). Most patients experiencing surgery move
forward uniquely when the epileptogenic tissue is resected and the surgical approach chosen
depends primarily on the localization and the degree of the epileptogenic zone, MRI findings,
the risk–benefit adjust of the respective surgery itself, and preoperative monitoring(Elger and
Schmidt, 2008).
The other non-pharmacological strategy of epilepsy management is vagus nerve stimulation

which includes discontinuous electrical stimulation of the vagus nerve through a pulse
6
generator device. It is utilized as an adjunct treatment for intractable partial seizures in adults
and children aged 12 years or older. Although the precise mechanism of anti-epileptic effect
of vagus nerve stimulation is unknown, change of synaptic activities within the thalamus,
changing neurochemistry within the solitary nucleus and modulation of the reticular
activating system are some of the hypothesized theories(Lim et al., 2018).
1.4.2. Pharmacological treatment

Right now, there are numerous AEDs utilized to treat epilepsy that act through distinctive
mechanism and include (i) increasing GABA-ergic action; (ii) inhibiting excitatory glutamate
neurotransmission; (iii) blocking sodium channels during high frequency discharges and (iv)
blocking voltage-dependent calcium channels (VDCCs)(Lasoń et al., 2011).
GABAergic drugs
In a wide range of animal models of epilepsy, drugs that enhance GABA mediated inhibition
are shown to have anti-convulsant activity and act through in one of the following ways; (i)
as GABA agonist (progabide);(ii)enhance synthesis or release of GABA (valproate and
vigabatrin)and (iii) through modulating channel opening(benzodiazepines and barbiturates)
(Treiman, 2001). Among barbiturates, phenobarbitone is active in almost all preclinical tests
used for screening AEDS and it inhibited tonic hind limb extension in MES test, PTZ
induced clonic seizure and amygdala kindling(Lasoń et al., 2011). The main indications for
barbiturates are generalized tonic-clonic seizures, partial seizures and drug resistant status
epilepticus (Hogg and Goodman, 2018).
Benzodiazepines are more effective in PTZ initiated seizure test than MES test. For example
clonazepam is extremely potent in PTZ test and nearly all of them have no impact in MES
induced seizure test(Lasoń et al., 2011). Benzodiazepines are mostly recommended for
treatment of status epilepticus(Hogg and Goodman, 2018). Vigabatrin, which is an
irreversible inhibitor of GABA transaminase is indicated as monotherapy for partial epilepsy
in adults whereas tiagabine which inhibits GABA transporter (GAT)-1 is indicated as add-on
therapy in partial and generalized drug resistant epilepsy(Deckers et al., 2000).
7
Calcium channel blockers
These groups of drugs are also indicated in the treatment of diverse sorts of seizures. In
general drugs that block T-type Ca+2 channels are effective in absence seizure, where as L-
type calcium channel blockers are effective against partial seizure(Ku³ak et al., 2004). Drugs
used for treatment of epilepsy by blocking calcium channels are ethosuximide and valproate.
Valproate is a broad-spectrum AED effective in the treatment of absence, myoclonic, focal,
and tonic-clonic seizures(Hogg and Goodman, 2018).
Sodium channel blockers
Sodium channel blockers have been the main pharmacological agents for management of
focal and generalized tonic-clonic seizure. They include drugs like phenytoin,
carbamazepine, lamotrigine, oxcarbazepine, rufinamide, lacosamide and eslicarbazepine
acetate. In addition to focal and generalized tonic-clonic seizure management, lamotrigine is
effective against absence seizure. Although phenytoin, carbamazepine, lamotrigine and
oxcarbazepine are more likely to cause idiosyncratic reactions, all these AEDs share similar
efficacy and adverse effect profile(Brodie, 2017).
Drugs that inhibit excitatory neurotransmission
Another class of AEDs target excitatory neurotransmission in the brain and includes
Felbamate and Falampanel. Felbamate has been shown to antagonize NMDA receptor and
also potentiates GABA mediated activities in hippocampus. The therapeutic use of felbamate
is limited because of its risk of aplastic anemia (Pellock et al., 2006, Pennell et al., 1995).
Talampanel is an allosteric inhibitor of AMPA receptor and has no activity at NMDA
receptor. In rodent models of epilepsy, the drug suppresses MES and PTZ initiated seizure
(Lasoń et al., 2011).
1.4.3. Herbal treatment

In addition to pharmacological treatment, plenty of medicinal plants have been used in
traditional medical practices to treat epilepsy across the globe. The use of medicinal plants to
8
treat epilepsy has been a common practice in the society of many countries like America,
India, Iran, China and Africa (Liu et al., 2017).
Several studies in developing countries have reported that large proportions of people with
epilepsy do not get treatment they need(Scott et al., 2001). Inspite of the fact that many
diagnosed and start on the treatment, they cease before long because of high cost of the
treatment, unavailability of AEDs and cultural beliefs(Radhakrishnan, 2009).Numerous
communities in Africa moreover associate epilepsy with evil spirits and superstitions,
encouraging treatment from traditional healers and religious leaders (Deresse and Shaweno,
2016). In Africa, up to 80% of the population use traditional medicine for primary health care
and the global market for herbal medicine is growing steadily (Muazu and Kaita, 2008).
Although numerous anti-epileptic drugs are accessible right now, they are associated with
severe, life threatening side effects(Sahranavard et al., 2014). Therefore, herbal medicines are
becoming the most useful approaches in the treatment of epileptic seizure in both developing
and developed countries reducing the complications caused by AEDs (Liu et al., 2017).
Numerous studies have demonstrated the anti-convulsant activity of several medicinal plants.
Among the many plants shown to have such activity in animal models, Moringa concanensis
(Moringaceae)(Joy et al., 2013),Prosopis cineraria Linn Druce(Mimosaceae)(Velmurugan et
al., 2012),Astragalus mongholicus(Leguminosae)(Aldarmaa et al., 2010), Lantana camara
Linn(verbanaceae)(Chinnala et al., 2013),Desmodium triflorum(Fabaceae)(Bhosle, 2013),
Colebrookea oppositifolia Smith(Lamiaceae)(Viswanatha et al., 2017), Sphaeranthus indicus
(Asteraceae)(Dighe and Barve, 2019),Punica granatum Linn(Punicaceae)(Das and Sarma,
2014), Indigofera tinctoria Linn(Fabaceae)(Garbhapu et al., 2011) and Vitex negundo Linn
(Verbenaceae)(Khokra et al., 2011) can be cited as examples.
1.5. Animal models of epilepsy
The investigation of potential helpful therapeutic agents for treatment of epilepsy requires the
utilization of seizure models. To meet the need for new drug development for treatment of
epilepsy, several experimental models were developed in which seizure activity is simulated
and different treatments are tested. Most of these models are seizure specific. Amongst the
many experimental seizure models, pentylenetetrazole (PTZ),maximal electroshock (MES),
9
kainic acid (KA) and strychnine are the foremost commonly used models in search for new
anti-convulsant drugs (Rubio et al., 2010).
PTZ is the most commonly employed seizure model which mirrors different forms of human
epilepsy. It is a chemo-convulsant which interferes with the action of GABA by specifically
binding to GABAA receptor, thereby blocking chloride conductance (Sayin et al., 1993).
Depending on the dose and routes of administration used, PTZ can simulate generalized
tonic-clonic seizure (grandmal) and absence seizure (petitmal) forms of human epilepsy.
Timed intravenous infusion test was used by scholars to determine the threshold dose of PTZ
used to induce the above types of epilepsy. It was reported that intravenous administration of
50mg/kg induces clonic seizure whereas 90mg/kg is used as a threshold dose for tonic-clonic
seizure (Rubio et al., 2010, De Deyn et al., 1992). Based on route of administration chosen,
PTZ is administered 15-30 min (Reddy and Rogawski, 2001), 30 min (Löscher et al., 1990)
or 60 min (Mandhane et al., 2007) following subcutaneous, intraperitoneal, and oral
administration, respectively, of standard drug or test compound to rodents. Following PTZ
administration, each animal is placed in individual plastic cage and are observed for
convulsive behavior(Chowdhury et al., 2013). The dose and route of PTZ administration
have been modified by different investigators (Vogel, 2002).
The major advantage of the model is its capacity to induce both petitmal and grandmal types
of seizure (generalized tonic-clonic seizure) based on the dose utilized i.e., graded doses of
PTZ can be administered to achieve wanted sort of seizure. Ineffectiveness of routine AEDs
like phenytoin, carbamazepine and oxcarbazepine against PTZ initiated seizure is the major
drawback related with the model(Ahmadiani et al., 2003, Velisek et al., 1992).
10
Maximal electro-shock seizure test is a test used to induce hind limb tonic extension through
bilateral corneal or transauricular electrical stimulation. This method is thought to be
predictive of anti-convulsant drug efficacy against grandmal (generalized tonic-clonic)
seizure. In this model an electric current of 50mA for mice and 150mA for rats is applied
through corneal or ear electrodes for 0.2 seconds. Upon application of stimulus, rodents
display five phases of convulsion: tonic limb flexion, tonic limb extension, clonic
convulsions, stupor and recovery or death. The test compound is said to possess anti-
convulsant activity, if it protects against the extensor stage of MES convulsion(Afrin et al.,
2017). The main advantage of the model is induction of generalized tonic-clonic seizure in
animals and plays an extraordinary part in searching drugs with numerous mechanisms of
activity. In addition, it is easy to perform the test and requires minimal technical
expertise(Castel-Branco et al., 2009). In MES test, animals receive supramaximal electric
current which is 5-10 times higher than the individual electrical seizure threshold of the
animals. Therefore, anti-convulsant compounds which can increase seizure threshold but not
potent enough to raise the seizure threshold above 50 mA for mice and 150mA for rats are
missed by this model(Löscher et al., 1991).
KA model of epilepsy is the most extensively studied model used to simulate TLE in
humans. KA is an agonist at kainate glutamate receptors and possesses potent convulsant
activity. Systemic administration of 12mg/kg i.p or s.c to rats induces convulsion that
originates in limbic structure, which then propagates to other brain areas. Studies revealed
that KA 20-40mg/kg (i.p) can be used to initiate convulsion in mice. Following i.p
administration of the convulsant, rodents display convulsive behavior such as wet-dog
shakes, staring, searching and gnawing, leading to hyperactivity, forelimb clonus and tonic-
clonic convulsions(Reddy and Kuruba, 2013).
KA model is very simple to be used, does not require sophisticated equipment and
regardless of its routes of administration can induce status epilepticus (SE). The major
impediment of the model is that, rats appear to show variable sensitivity based on strain,
age, sex and weight. In addition, KA is a direct excitotoxic and this makes troublesome to
distinguish the neuronal damage whether it is due to the direct excitotoxicity or seizure
induced neuronal damage(Ben-Ari et al., 1981).
11
Strychnine is a competitive inhibitor at all glycine receptors, which is an important
inhibitory neurotransmitter in the spinal cord. In this model, test compound or standard drug
(diazepam 5 mg/kg) is administered to rodents 1h before administration of strychnine at a
dose of 1.75-2.5mg/kg i.p. Occurrence of tonic extensor convulsions and death is observed
during a 1 h period(Hunter et al., 1989, Vogel, 2002). An advantage of strychnine as a
convulsant chemical is its capacity to be applied either topically or systemically (Zabara,
1992). Invasiveness (application of surgical procedures) , potential impact of anesthetic
agents used on behavioral analysis of the animals amid the experiment and difficulty of
exact application of the systemically managed convulsant to particular location of the brain
is its major confinements (McCandless and FineSmith, 1992).
In addition to the most commonly used seizure models described above, amygdala
kindling(Löscher, 2017),pilocarpine(Reddy and Kuruba, 2013), picrotoxin(Kesim et al.,
2012) and penicillin(Bostanci and Bağirici, 2007) can also be used to initiate seizure
induction in laboratory animals during search for potential anti-convulsant agents.
1.6. Overview of the experimental plant

The genus Ajuga is a member of the Lamiaceae family that contains more than 100 species
and 50 sub-species distributed over the world(Topçu et al., 2004).It is traditionally used for
treatment of distinctive sicknesses and some of them were tested for the specified activity.
The most common ailments treated by the genus Ajuga include cancer(Pal et al.,
2014),depression(Kayani et al., 2016),inflammation(Gautam et al., 2011),diabetes and
hypertension(Tahraoui et al., 2007, Boudjelal et al., 2015, El-Hilaly et al.,
2021),Alzheimer’s disease(AD)(Movahhedin et al., 2016),wound healing(Khalil et al.,
2007),pain(anti-nociceptive)(Khanavi et al., 2014),bacterial infections (Rahman et al., 2013,
Setif, 2011),helminths infestation and dental problems(Rahman et al., 2016).
Ajuga integrifolia Buch.Ham ex D.Don (synonyms: Ajuga remota Benth, Ajuga bracteosa
Wall ex Benth.) (Figure-1) is a shrub that grows broadly in East Africa at an altitude of
1500-3400 m above sea level. It also grows in Saudi Arabia, Yemen , Afghanistan and East
Asia (Tafesse et al., 2017) . In Ethiopia, it grows in different parts of the country including
12
Bale, Gojam, Gondar, Hararghe, Kefa, Shoa, Sidamo, Tigary, Wollo and Dawuro (Seifu,
2017).
A.integrifolia is a herb covered with short hairs and its stem can grow up to 40 cm high. Its
leaves are oblanceolate and coarsely toothed and it flowers from late August to October
(Seifu, 2017). In Ethiopia, A.integrifolia is known by different vernacular names like
Armagusa (Afan Oromo) (Kefalew et al., 2015), Akorarach or Tut astil (Amharic) (Degu et
al., 2020), Anamuro (Guragegna) (Teka et al., 2020a) and Anamuro (Sidamigna) (Tefera
and Kim, 2019).
Ethno-botanical studies conducted in different parts of the world revealed that A. integrifolia
is utilized for treatment of numerous conditions. For example, in Himalaya’s folk medicine,
the leaves are used for edema, febrile conditions ,gout, rheumatism and amenorrhea(Singh
and Thakur, 2014). In India, one cup of decocted root assorted with honey is administered
orally after breakfast to treat malaria (Wangpan et al., 2016). In Kenyan traditional medicine
it is utilized for treatment of diabetes (Keter and Mutiso, 2012) and malaria (Njoroge and
Bussmann, 2006).
In Ethiopian folk medicine, the plant is used for the treatment of diabetes(Meresa et al.,
2017),retained placenta(Giday et al., 2009), malaria(Asnake et al., 2016, Gedif and Hahn,
2003), stomachache(Regassa, 2013, Tilahun, 2018),wound and ameobiasis(Giday et al.,
2010),cancer(Tesfaye et al., 2020), diarrhea(Parvez and Yadav, 2010) , liver problem(Teka
et al., 2020b), anthrax(Mesfin et al., 2014),hypertension(Getahun, 1976, Teshome et al.,
2019), pneumonia(Regassa et al., 2017) and epilepsy(Abera, 2014, Atnafu et al., 2018). The
plant parts used for the specified activities are leaves, stem and root (Tafesse et al., 2017).
Some of these traditional uses and the lethal dose (LD50) of the plant have been investigated
by different scholars. The acute toxicity study conducted on the aqueous and hydro-
alcoholic leaf extract of A. integrifolia demonstrated that the LD50 of the plant is greater
than 5000mg/kg as the animals experienced no signs of overt toxicity at this dose. It was
also reported that leaf extracts have anti-hypertensive(Hailu and Engidawork, 2014), anti-
malarial(Nardos and Makonnen, 2017), anti-type I and II HIV(Asres et al., 2001) and anti-
Mycobacterium tuberculosis activity (Cantrell et al., 1999). There are also reports that
13
confirmed anti-epileptic activity of the leaf(Getaneh, 2020) and stem(Qasim et al., 2017)
extracts. In addition to the leaf, different extracts of the root of the plant was also reported to
possess anti-diabetic activity (Alene et al., 2020, Tafesse et al., 2017).
The leaf of the plant was found to contain different secondary metabolites such as phenols
,flavonoids ,alkaloids ,terpenoids ,carbohydrates and steroids(Tebeje, 2019) .
In addition to the secondary metabolites listed above, the leaf also contains essential oil such
as limonene, α-humulene, β-myrcene, elemol, camphene, β-caryophellene, and α-
phellendrene(Vohra and Kaur, 2011). From aerial parts (leaf and stem) of the plant different
diterpenes such as ajugarin I, ajugarin II, ajugarin IV, ajugarin V and ajugapitin were
isolated using high performance liquid chromatography(Coll and Tandrón, 2005).
Triterpene, such as, ergosterol-5,8-endoperoxide, with anti-mycobacterium tuberculosis
activity was also isolated from the aerial parts (Cantrell et al., 1999). The root of the plant
was also investigated and found to contain secondary metabolites such as alkaloids,
flavonoids, phenols, glycosides, terpenoids, tannins, saponins and steroids. The bio-active
substances found in different parts of the plant might play a predominant role in the
effectiveness of the plant in many conditions (Bekeri et al., 2018).
14
Figure 1: Photograph of Ajuga integrifolia Buch.Ham ex D.Don (Photo Credit: Tesfaye
Desalegn)
15
1.7. Rationale of the study
Epilepsy is one of the foremost common neurological disorders, especially in poor zones of
the world, and can have a devastating impact on individuals with the disorder and their
families. Its burden in low income countries is two-fold more than observed in high income
countries(Newton and Garcia, 2012). Epilepsy represents around 0.7% of the overall global
burden of diseases measured in Disability adjusted life years (DALYs) and ranks as the 36th
leading cause of DALYs globally. Pharmacological treatment is the mainstay of therapy in
the management of epilepsy and about 70% of the cases are controlled by currently available
AEDs(Espinosa-Jovel et al., 2018). However, poor availability and high cost of the
medications are the major problems associated with the conventional AEDs (Randrianarivo
et al., 2016) and about half of epileptic patients treated with these drugs develop at least one
adverse effect during their treatment with AEDs(de Biase et al., 2019). In addition, although
the AEDs can be used for controlling epilepsy in larger percent of the patients, most of them
do not prevent or reverse the pathological processes that underlie the disease and 30–40% of
patients typically develop pharmacoresistant epilepsy(M Manchishi, 2018, Prunetti and
Perucca, 2011).
Many studies have reported that herbal medicines are commonly used for treatment of
epilepsy because AEDs fail to control seizure in 30% of the epileptic patients. In addition,
patients also take herbal medicine because of economic and cultural factors(Liu et al., 2017).
Hence, continuing search for new therapies of plant origin with fewer side effects and better
efficacy is important.
Ethno-Botanical studies conducted in many parts of Ethiopia showed that use of herbal
medicine for management of epilepsy is also a common practice(Abera, 2014, Atnafu et al.,
2018). Although a recent experimental study reported anti-epileptic activity of A.
integrifolia(Getaneh, 2020);it was limited to acute models and did not quantify the major
phytoconstituents. Thus, further studies that bridge theses gaps are required. Hence the
present study was initiated to assess the efficacy of the plant in both acute and chronic
models as well as quantify major constituents thought to be responsible for the anti-
convulsant effect. On this regard, the results of this study could serve as baseline
16
information for the development of new anti-epileptic drug for pharmacoresistant epilepsy
and can give a clue for isolation and identification of active principal that can be used as a
lead compound.
17
2. OBJECTIVES
2.1. General objective

➢ To verify the anti-convulsant activity of the leaf Soxhlet extracts of A.integrifolia
in mice and quantify the major secondary metabolites.
2.2. Specific objectives

✓ To assess anti- convulsant activity of the extracts in PTZ- induced seizure test.
✓ To determine the effect of the extract in MES- induced seizure test.
✓ To evaluate the effect of the extract in PTZ- induced kindling model
✓ To quantify the major secondary metabolites (flavonoids, phenols and alkaloids)
content of the plant.
18
3. EXPERIMENTAL METHODS
3.1. Drugs and chemicals

Pentylenetetrazole(Sigma Aldrich, Germany), gallic acid(Merck, Germany)and Folin Cio-
calteu reagent was obtained from the Department of Pharmacology and Clinical Pharmacy.
NaOH(Loba Chemie, India),AlCl3(Loba Chemie, India),Chloroform(Loba Chemie, India),
HCl (BDH laboratory supplies, England),citric acid(Avonchem,UK),Na2HPO4(BDH
laboratory supplies, England), Atropine(BDH chemicals, England),Quercetin Dihydrate
(Sigma Aldrich, Germany) were obtained from the Department of Pharmacognosy and
Pharmaceutical chemistry.Tween80(Loba chemie,India), BCG(Sisco Chemical laboratories,
India),n-hexane(Loba Chemie, India), ethyl acetate (Trust chemical laboratories, UK),
methanol(Sisco Research Laboratories, India),Normal saline solution(Sansheng
pharmaceuticals, Ethiopia), potassium acetate (Blulux, India), Sodium valproate (Sanofi,
Spain) and Phenytoin(Macleods Pharmaceuticals, India),were obtained from their respective
vendors. All the drugs and chemicals used were of analytical grade.
3.2. Experimental Animals

Healthy male Swiss albino mice (8-10 weeks, 22-28g) were used for the current study. The
animals were obtained from the Ethiopian Public Health Institute as well as, the animal unit
of School of Pharmacy, College of Health Sciences, Addis Ababa University, Addis Ababa,
Ethiopia. The animals were housed in group of six and acclimatized to laboratory conditions
for a week before starting of the experiment. The mice were kept under standard
environmental condition (12h light/ dark cycle) and provided with a commercial food pellet
and water ad libtum. All procedures and techniques used in this study were conducted in
accordance with the National Institute of Health Guidelines for the Care and Use of
Laboratory Animals (Council, 2010). The protocol was approved by IRB of the School of
Pharmacy on December 03, 2019 with approval number of RRB/SOP/199/2019.
3.3. Collection of plant material

Leaves of A.integrifolia were collected from its natural habitat in Girar Jarso district located
180 km north of Addis Ababa, Oromia region, North Ethiopia. Collected plant specimen
was identified & authenticated by a taxonomist, Mr. Melaku Wondafrash at the National
19
Herbarium, College of Natural and Computational Sciences, Addis Ababa University and a
voucher specimen (TD001) was deposited for future reference.
3.4. Preparation of the extracts

The collected leaves were washed thoroughly with tap water to remove dirt and soil and
shade dried at room temperature for three weeks. The air dried leaves were subjected to size
reduction using mechanical grinder to get a coarse powder. The powdered plant material
(500g) was extracted by successive Soxhlet extractor (Pyrexquickfit, UK) using 4 liters of
each of the following solvents; n-hexane, ethyl acetate and methanol. After extracting with
the above solvents, the remaining residue was macerated with one liter of distilled water
three times for 72 h with occasional shaking to get the residual aqueous extract. Each time
before extracting with the next solvent, the powdered plant material was air dried over night.
The resulting solution was first filtered by cotton gauze for aqueous extract and later by
Whatman filter paper (No.1) for all solvent extracts. The non-aqueous filtrates were
concentrated in a rotary evaporator (Buchi, Switzerland) under reduced pressure at 40 °C
and the water extract was freeze dried by using a lyophilizer( Korea vacuum limited, Korea)
to obtain the respective extracts. The yield (w/w) in terms of dry material for n-hexane, ethyl
acetate, methanol and water was 2.95%, 4.4%, 15.45%, and 3.29% respectively. The dried
extracts were kept in a refrigerator at -20°C until use.
3.5. Grouping and dosing of animals

The animals were randomly assigned in to five groups for each solvent extracts, each group
containing six mice. In the acute model: group I served as negative control and treated with
the vehicle used for reconstitution (2% Tween80 for the non-aqueous extracts and distilled
water for the aqueous extract). Group II was a positive control and treated with sodium
valproate 200 mg/kg (SV200) for PTZ model and Phenytoin 25 mg/kg (PHY25) for MES.
Group III-IV were test groups and administered with 100mg/kg, 200mg/kg and 400mg/kg
doses of the respective extracts. The most active extract (ethyl acetate extract) was
investigated in the chronic model of epilepsy (PTZ kindling) using the same grouping
(SV200 was used as standard). All animals were administered orally and the maximum
volume used was 10ml/kg. The doses were determined by a pilot study conducted before
commencement of the experiment.
20
3.6. Anti-convulsant activity test
Anti-convulsant activity of the plant was evaluated using acute and chronic models of
epileptic seizures.
3.6.1. PTZ induced seizure

For this test, a method modified by (Salem et al., 2019) was used. The mice in each group
received different doses of extracts, standard drug and vehicle. After 60 min of oral
administration as described in section 3.5, freshly prepared PTZ in normal saline (80mg/kg)
was administered to the scruff of the neck of each mouse. The animals were then placed in a
transparent cage and observed for convulsive behavior for 30 min using a video recording.
Fore limb or hind limb clonic seizure (Figure 2) was taken as endpoint. The latency to clonic
convulsion and percentage protection of mortality were recorded and compared with
negative controls. Percentage protection of mortality was calculated as follows.
No of death in control− No of death in test/standard x100

Percentage protection of mortality= Number of death in control
Figure 2: Clonic seizure exhibited after 80mg/kg subcutaneous injection of PTZ.
21
3.6.2. MES induced seizure
For this experiment a method described by (Raza et al., 2001) was used. After an hour of
oral administration as described in section 3.5, seizure was induced by auricular stimulation
(50mA, 150 Hz, 0.2 sec) using an electro-convulsometer (Rolex Ambala, India).The ear clip
electrodes were moistened with normal saline before application for better conductance.
Each animal was closely followed for 2 min using a video recorder. Upon exposure to
electric current, the animals exhibited various phases of tonic-clonic seizure that include
immediate short-lived flexion of fore limbs followed by extension of hind limbs. After the
end of the extensor phase, they showed stupor phase that finally led to recovery or death.
Duration of HLTE (i.e. outstretching of the animals 180 ° to the body axis) and protection
against mortality were recorded.
Percentage protection of mortality was calculated as follows:
No of death in control− No of death in test/standard x100

Percentage protection of mortality=
Number of death in control
In this experiment reduction in the mean duration of HLTE of MES convulsion was
considered as having anti-convulsant activity(Mahendran et al., 2011).
3.6.3. PTZ induced Kindling

One hour after administration as described in section 3.5, mice were kindled with repeated
(every 48 h) intraperitoneal administration of freshly prepared PTZ (35mg/kg) for 13 days.
On each day animals were closely observed for 30 min using a video recorder after PTZ
injection to measure intensity of seizure. The following seizure scores were used to identify
fully kindled mice: Stage 0(no response); stage 1(hyperactivity, ear and facial twitching);
Stage 2(head nodding and myoclonic body jerks); stage 3(forelimb clonic seizure); stage
4(generalized clonic seizure with falling) and stage 5(generalized tonic-clonic seizures).
Animals that showed at least three consecutive stage 4 or stage 5 seizure score were thought
to be kindled. Animals that did not show three consecutive stage 4 or 5 were considered to
be protected (Abdel-Zaher et al., 2017).
22
3.7. Quantification of phytochemical constituents
3.7.1. Quantification of total flavonoids content

Total flavonoids content (TFC) was estimated as described by(Chang et al., 2002) with
slight modification. Ten mg of the ethyl acetate extract (EA) was dissolved in methanol to
prepare a stock solution of 1mg/ml. One ml of the stock solution was transferred to a test
tube and mixed with 0.1ml of 10% aluminum chloride, 0.1ml of 1M potassium acetate and
2.8ml of distilled water. The mixture formed was allowed to stand for 30 min at ambient
temperature, after which absorbance was recorded at 415 nm using a UV-spectrophotometer
(Jenway Model 6500, England). Quercetin was used to construct the standard curve. It was
dissolved in methanol and a serial dilution was used to prepare 1.5625, 3.125, 6.25, 12.5 and
25µg/ml standard solution. The same procedure was followed to prepare the blank solution.
All experiments were performed in triplicate. The TFC of the extract was expressed as mg
of quercetin equivalent per gram of the dry weight of the extract and calculated as follows;
𝑇𝐹𝐶 = CV/M
Where C is the sample concentration (µg/ml) from the calibration curve, V is the volume of
methanol used to dissolve the extract and M represents the mass of the dry extract used in
gram.
3.7.2. Quantification of total phenolic content

Folin-Ciocalteu reagent was utilized for this assay. Folin reagent (1ml of 2N) was diluted
with 20ml of distilled water. To determine the total phenolic content (TPC), 1ml of the
prepared EA solution (250µg/ml) was transferred to a test tube and 0.5ml of Folin reagent
was added and allowed to stand for 8 minutes. Thereafter 2ml of 7.5% sodium carbonate in
distilled water was mixed with the solution and incubated for 30 min at ambient
temperature. An absorbance was later recorded at 765nm by a UV-spectrophotometer and
compared with gallic acid calibration curve. Gallic acid was prepared in different
concentration of 3.125, 6.25, 12.5, and 25µg/ml by serial dilution to draw the standard
curve. The same procedure was followed to prepare gallic acid and the blank solution. All
experiments were conducted in triplicates and the average value was taken. The TPC was
described as gallic acid equivalent per gram of sample(Maria et al., 2018).
23
3.6.3. Quantification of total alkaloids
Total alkaloid content (TAC) of the plant was determined according to a method described
by (Tabasum et al., 2016) with slight modification. The reaction that took place between
alkaloid and bromocresol green (BCG) was used to quantify the total alkaloid content by
spectrophotometric method. Accordingly, 2ml of plant extract in methanol (1mg/ml) was
dissolved in 2ml of 2N HCl and filtered. One ml of this solution was transferred to a
separating funnel and washed with 5ml of chloroform twice. pH of the solution was adjusted
to neutral by adding 0.1N NaOH. After pH adjustment, 5ml of BCG solution (prepared by
heating 69.8 mg of BCG with 3ml of 2 N NaOH and 5ml of distilled water and then diluted
to 1000ml with distilled water) along with 5ml of phosphate buffer (prepared by adjusting
the PH of 2M sodium phosphate (71.6 gm of Na2HPO4 in 1L distilled water) to 4.7 with 0.2
M citric acid (42.02 gm) citric acid in 1 L distilled water) was added. The aggregate formed
was shaken and the mixture formed was extracted with 5ml of chloroform twice by vigorous
shaking. The extract was collected in 10ml volumetric flask and the volume was made up to
10ml with chloroform. The absorbance of the mixture in chloroform was measured at
470nm by using a UV-spectrophotometer. For standard curve construction, atropine was
dissolved in methanol to prepare different concentrations (0.5, 0.25, 0.125, 0.062 and
0.03125 mg/ml) of the standard solution. One ml of this solution was taken and transferred
to a separating funnel and 5ml of phosphate buffer along with 5ml BCG solution was added
and shaken gently with 5ml of chloroform twice. The mixtures formed was collected in
10ml volumetric flask and diluted to the volume with chloroform and absorbance was
measured at 470nm. The blank was prepared as described without atropine. The assay was
run in triplicates and the average value was taken.
3.8. Data Analysis

All experimental data are expressed as mean ± SEM and subjected to statistical analysis by
SPSS windows version 25 statistical packages. Statistical analysis of the difference among
groups was performed with One way analysis of variance (ANOVA) followed by Tukey’s
post-hoc test. For PTZ kindling, two-way analysis of variance and Benferroni’s post hoc test
was used for multiple comparison of the mean difference between the groups. The analyses
were performed with 95% confidence interval and the significance was set at p<0.05.
24
4. RESULTS
4.1. Anti-convulsant activity in PTZ induced seizure

All A.integrifolia leaf solvent extracts but the aqueous extract (AA) possessed anti-
convulsant activity against absence (petitmal) seizure model of epilepsy as evidenced by an
increased mean latency to clonic convulsion (Table 1). EA was the most effective extract, as
it prolonged the mean onset of clonus and decreased percent mortality better than the other
extracts. The mean latency to clonic seizure was significantly increased (p<0.01) with all
doses of EA compared to controls, with maximum effect (13.17min) achieved by EA
400mg/kg (EA400). The increment in mean onset of clonus was in a dose dependent manner
as EA400 significantly delayed onset of clonus compared to EA200 mg/kg (EA200)
(p<0.01) and EA100 mg/kg (EA100) (p<0.001). EA also decreased percent mortality in a
dose- dependent manner, with maximum protection (66.67%) conferred by EA 400 (Table
1)
The hexane extract (HA) also significantly increased mean latency to clonic seizure
compared to control, with a maximum increase (12.67min, p<0.001) displayed by HA400
mg/kg (HA400). The effect produced by HA400 was significantly greater than the one
produced by HA 100 mg/kg (HA100) (p<0.001) and HA200mg/kg (HA200) (p<0.01). As
regards to mortality, whilst HA200 and HA400 were able to decrease death by 33.33% and
50%, respectively, HA100 was devoid of any effect (Table 1).
The methanol extract (MA) at 100 mg/kg (M100) was not effective in both parameters of
this experimental paradigm. However, the MA, at dose of 200mg/kg (MA200) and
400mg/kg (MA400) significantly increased (p<0.001) mean latency and fairly well protected
mortality compared to controls. By contrast, the AA was devoid of any effect at all doses
(Table 1).
The standard drug used (SV) was superior in both measures, as it significantly increased
latency (p<0.01) and decreased mortality (83.33%) compared to all doses of the extracts
used in this experiment.
25
Table 1. Anti-convulsant activity of the Soxhlet leaf extracts of Ajuga integrifolia in
pentylenetetrazole induced seizure
Group Mean latency to clonic Percentage protection

seizure (min) of mortality
CTN 3.00±0.447 -
EA100 6.33±0.333a2b3d3e3 33.33
EA200 10.17±0.543a3b3c3e2 50.00
EA400 13.17±0.703a3b2c3d2 66.67
SV200 16.67±0.494a3 83.33
CTN 3.00±0.447 -
HA100 5.17±0.307a1b3d3e3 0.00
HA200 9.33±0.494a3b3c3e2 33.33
HA400 12.67±0.667a3b3d2 50.00
SVP200 16.67±0.494a3 83.3
CTN 3.00±0.447 -
MA100 4.84±0.477b3d3e3 0.00
MA200 7.50±0.619a3b3c2e2 16.67
MA400 10.00±0.577a3b3c3d2 33.33
SV200 16.67±0.494a3 83.33
DWC 2.50±0.342 -
AA100 3.00±0.365 0.00
AA200 3.33±0.333 16.67
AA400 3.50±0.428 16.67
SV200 16.67±0.494a3c3d3e3 83.33
Values are expressed as mean ±SEM. (n=6 mice) acompared to control, b
compared to sodium valproate, c
compared to 100mg/kg, d compared to 200mg/kg, e compared to 400mg/kg. 1 p<0.05, 2 p<0.01, 3 p<0.001. CTN:
group treated with 2% tween80, CDW: group treated with distilled water, SV: sodium valproate, EA: Ethyl
acetate extract of Ajuga integrifolia, HA: hexane extract of Ajuga integrifolia, MA: methanol extract of Ajuga
integrifolia, AA: aqueous extract of Ajuga integrifolia. Numbers refer to doses in mg/kg.
26
4.2. Anti-convulsant activity in MES induced seizure
As it can be observed from Table 2, all leaf solvent extracts of A.integrifolia except the
aqueous extract (AA) produced variable results in reducing the mean duration of HLTE and
percent mortality in MES test. The EA extract was the most effective extract, as it reduced
the duration of HLTE and percent mortality better than the other extracts. The mean duration
of HLTE was significantly decreased (p<0.001) with all doses of EA compared to controls,
with maximum reduction (6.33sec) achieved by EA400. The reduction in mean duration of
HLTE was in a dose dependent manner as EA400 significantly decreased the duration of
HLTE compared to EA200 (p<0.01) and EA100 (p<0.001). EA also decreased percent
mortality, with maximum protection (50%) achieved by EA200 and EA400 (Table 2).
The hexane extract (HA) also significantly decreased mean duration of HLTE compared to
control, with maximum reduction (8.17 sec, p<0.001) obtained by HA400. The effect
displayed by HA400 was significantly greater than the one produced by HA100 (p<0.05)
and HA 200mg/kg (HA200) (p<0.001). Concerning mortality, HA200 and HA400 decreased
death by 16.67% and 33.33%, respectively, while HA100 was devoid of any effect.
MA100 was not effective in both parameters used to evaluate anti-convulsant activity of the
plant in MEs test. However, MA200 and MA400 significantly reduced (p<0.001) the mean
duration of HLTE and equally resulted in percent protection of mortality (16.67%)
compared to controls. By contrast, the AA was devoid of any effect at all doses (Table 2).
The standard drug used (PHY) was superior in both measures as it significantly abolished
the occurrence of HLTE (p<0.001) and decreased mortality (83.33%) compared to all doses
of the extracts used in this experiment.
27
Table 2. Anti-convulsant activity of the Soxhlet leaf extracts of Ajuga integrifolia in MES
induced seizure.
Group Mean duration of HLTE(sec) Percentage protection

from mortality
CTN 18.50±0.563 -
EA100 14.33±0.964a3b3d3e3 33.33
EA200 9.67±0.715a3b3c3e2 50.00
EA400 6.33±0.422a3b3c3d2 50.00
PHY25 0.00a3 83.33
CTN 18.50±0.563 -
HA100 16.67±0.558a1b3d2e3 0.00
HA200 13.00±0.516a3b3c2e3 16.67
HA400 8.17±0.477a3b3 33.33
PHY25 0.00a3 83.33
CTN 18.50±0.563 -
MA100 16.50±0.671b3d1e3 0.00
MA200 14.00±0.73a3b3e3 16.67
MA400 10.17±0.307a3d3 16.67
PHY25 0.00a3 83.33
DWC 19.83±0.307 -
AA100 19.33±0.615 0.00
AA200 18.33±0.422 16.67
AA400 17.83±0.477 16.67
PHY25 0.00a3 83.33
Values are expressed as mean ±SEM. (n=6 mice), a compared to control, b compared to Phenytoin, c compared
to 100mg/kg, d compared to 200mg/kg, e compared to 400mg/kg. 1 p<0.05, 2 p<0.01, 3 p<0.001. CTN: group
treated with 2% tween80, CDW: group treated with distilled water, PHY: phenytoin, EA: Ethyl acetate extract
of Ajuga integrifolia, HA: hexane extract of Ajuga integrifolia, MA; methanol extract of Ajuga integrifolia,
AA: aqueous extract of Ajuga integrifolia.. Numbers refer to doses in mg/kg.
28
4.3. Anti-convulsant activity in PTZ kindling Model
The most active extract (EA) in the acute seizure model also showed anti-convulsant activity
in the chronic seizure model (Figure 3). For this test, 13 injections of PTZ (35 mg/kg i.p) on
alternate day basis produced full kindling starting from the 11th -13th injections in controls,
while treatment of mice with different doses of EA extract produced variable effects. EA400
significantly protected (p<0.001) the animals from developing consecutive stage 4 and/ or 5
seizure on the last three injections compared to controls, which indicated protection of the
animals. Likewise, EA200 also produced significant effect (p<0.01) in protection of kindling
compared to controls. On the other hand, EA100 was ineffective in the parameter used in
this experimental model. The standard drug used (SV) was superior in the parameter used as
it significantly prevented (p<0.001) occurrence of kindling compared to controls.
As it can be observed from Figure 3, the EA extract reduced mean seizure stage in a dose
dependent manner. Statistical analysis revealed that all doses of the extract and the standard
drug (SV) significantly reduced (p<0.05) the seizure stage on the first injection compared to
controls. On the second injection, the seizure score observed was significant for all doses of
the extract (p<0.01) and the standard (p<0.001) compared to the negative control but no
significant difference was still seen between extract and SV pretreated group. Difference in
stage of seizure after PTZ injection between treatment groups was observed from the third
injection onwards.
As the number of injection increased, significant difference in mean seizure stage between
EA100 and negative control group started to disappear. Indeed no detectable difference in
mean seizure stage was noted between the two groups from the 9th -13th injections. This
indicated failure of EA100 to protect the animals from PTZ induced kindling. EA400
pretreated group started to respond to the chemoconvulsant PTZ from the 6th injection but no
significant effect was seen between EA400 and SV200 until the 9th injection.
29
5
4
** NC
** **
seizure stage
3 EA100
EA200
***
*** *** EA400
2
SV200
1
0 *** *** ***

0 2 4 6 8 10 12 14
number of injection
Figure 3: Effect of Ethyl acetate extract on Pentylenetetrazole induced kindling in mice.

Animals were treated with 100mg/kg, 200mg/kg and 400mg/kg ethyl acetate extract and
200mg/kg sodium valproate along with PTZ 35mg/kg i.p every other day for 13 days. Data
expressed as mean± SEM of 22 days observations. ** p<0.01, *** p<0.001.
4.4. Quantification of Secondary Metabolites
4.4.1. Determination of Total flavonoids content

The assay performed to determine TFC using the standard produced a curve with a
regression coefficient (R2) =0.9964, slope (m) =0.02432 and y-intercept= 0.0159 (Figure 4).
The extract was found to contain 9.045 ±0.8445 mg of quercetin equivalent (QE) of
flavonoids per gram of dry extract used.
30
0.8
R 2 = 0.9964
absorbance(415)
0.6
9
− 0.015
x
0.4
.0 2432
y = 0
0.2
0.0
0 10 20 30
concentration(g/ml)
Figure 4: Standard curve constructed for quercetin based on absorbance measured at 415
nm. Data are expressed as mean ±SEM, n=3.
4.4.2. Determination of total phenolic content
The experimental study conducted to determine the TPC using gallic acid produced a
standard curve with (R2)= 0.9982, slope(m)=0.009323 and y-intercept=0.005289 (Figure 5).
The EA extract was found to contain 21.928 ±1.118 mg of gallic acid equivalent (GAE) per
gram of dry extract utilized.
31
0.4
absorbance (765nm)
R 2 = 0.9982
0.3
0.05289
0.2 9323x +
y = 0.00
0.1
0.0
0 10 20 30
Figure 5: Standard curve constructed for gallic acid based on absorbance measured at 765
4.4. 3. Quantification of Total alkaloid content
The assay conducted to determine TAC of the EA extract using atropine produced a
standard curve with (R2) =0.9938, slope (m) =0.002625 and y-intercept=0.001750 (Figure
6). The extract was found to contain 10.002±0.119 mg atropine equivalent per gram of dray
extract used.
32
0.15
absorbance (470 nm)

R 2 = 0.9938
0.10 0 1 750
0
x + 0.
0 2 625
0
y = 0.
0.05
0.00
0 20 40 60
Figure 6: Standard curve constructed for atropine based on absorbance measured at 470
33
5. DISCUSSION
In Ethiopian traditional medicine, the dried leaf of A.integrifolia is pounded and mixed with
nut oil and the patient is informed to eat it to treat epilepsy. The dried leaves of the plant
were therefore extracted with organic solvents of different polarity and oral dosing of the
extract was used to replicate the traditional use.
Male mice were used for the anti-convulsant activity testing because female mice are less
sensitive to the convulsive activity of MES and PTZ due to the effect of estrus cycle on
seizure threshold (Medina et al., 2001, Dai et al., 2014, Tandon and Gupta, 2005, Kaboutari
et al., 2012).
MES and PTZ which were developed long years ago have been used as a cornerstone in the
early search of potential anti-convulsant drugs. Since their development, many compounds
have been tested against MES and PTZ in laboratory animals for anti-epileptic activity.
These two models are the most commonly employed seizure models because they are rapid
and easy to perform for the preliminary screening of potential anti-convulsant drugs
(Swinyard, 1949).
In the present study, solvent leaf extracts of A.integrifolia were found to possess anti-
convulsant activity in both acute and chronic seizure models that appeared to vary with dose
and nature of the extract. EA extract produced the highest effect in both parameters used in
the acute PTZ experimental paradigm among all solvent extracts. The anti-convulsant
activity demonstrated by the EA extract could be attributed to the presence of secondary
metabolites such as flavonoids, phenols, alkaloids, terpenoids and steroids, which were
reported by earlier studies(Tebeje, 2019) as well as by the present study. A similar type of
study done on crude stem extract and solvent fractions of A.integrifolia reported anti-
convulsant activity of the plant in PTZ model of seizure. Although ethyl acetate fraction was
the most effective among all fractions, the effect produced in terms of increasing mean onset
of clonic seizure by the higher dose of the fraction (EA 1000mg/kg,7.875 min)(Qasim et al.,
2017) was lower than the one produced by the most effective extract(EA400mg/kg,
13.17min) of the current study. The better anti-convulsant activity obtained by the present
34
study could be because of better accumulation of secondary metabolites such as terpenoids
in leaves than stem and the difference in geographical location(Li et al., 2020)
The hexane extract ranked next to EA extract in increasing the mean latency to clonic
seizure and percent protection of mortality in PTZ induced seizure test. In line with the test
result, the anti-convulsant activity displayed by HA in terms of the parameters used, was
lower than the one displayed by the EA extract. It was reported that the leaf hexane fraction
of A.integrifolia did not contain the major phytoconstituents such as phenols and
flavonoids(Dechasa, 2019). Therefore, the reduced anti-convulsant activity of the extract
could be because of the absence of the indicated phytoconstituents.
Methanol extract showed the least anti-convulsant activity among all solvent extracts. The
lowest dose of the extract was unsuccessful in delaying the mean onset of clonus and
protecting the animals from death though the medium and highest dose displayed anti-
convulsant activity. The absence of steroids in the methanol extract, as it was reported,
might contribute for the reduced anti-convulsant activity(Tebeje, 2019). At any dose the
aqueous extract did not produce a considerable effect in delaying onset of clonic seizure and
increasing percent protection of mortality. This might because of reduced concentration of
secondary metabolites in aqueous extract as studies showed the yield of secondary
metabolites such as alkaloids, phenols and flavonoids increases with non-polar solvents than
polar solvents(Bouterfas et al., 2016).
A previous similar type of study done on the leaf crude extract and solvent fractions of the
plant collected from different geographical location, Ghimbi district, reported anti-
convulsant activity of A.integrifolia in PTZ model(Getaneh, 2020). In this study, the mean
latency to clonic seizure exhibited by the most active solvent fraction (butanol 400 mg/kg)
was greater (15.51min) than the most active extract (EA 400 mg/kg, 13.17min) in the
current study. The subtle difference might have emanated from variation in the geographical
location(Borges et al., 2017).
35
The present study also evaluated anti-convulsant activity of the plant in MES model. Once
again, EA extract displayed the highest reduction in the mean duration of HLTE than HA
and MA extracts which might be because of accumulation of sufficient concentration of the
phytoconstituents in the EA extract. Like in the PTZ test, the AA extract did not produce an
appreciable effect in reducing the mean duration of HLTE may be because of absence of
active metabolites.
It was reported that crude leaf extract and solvent fractions A.integrifolia possessed anti-
convulsant activity in MES test(Getaneh, 2020). However, the highest dose of the most
active extract of the current study (EA400 mg/kg) produced a significant effect in reducing
the mean duration of HLTE (6.33 sec) compared to the one displayed by its corresponding
reported butanol fraction (400 mg/kg, 8.33 sec). The difference in the effect produced could
emanate from the type of extract used indicating less non-polar components were
responsible for the anti-convulsant activity of the plant in MES test.
MES and PTZ, which are only models for acute seizure, cannot simulate chronic
dysfunction of brain which is seen in epilepsy and cannot be used for discovery of potential
AEDs for pharmacoresistant epilepsy. Therefore, kindling is widely employed to study the
process of epileptogenesis and anti-epileptic drug discovery(Löscher, 2002). Thus the
present study also investigated the effect of the most active extract (EA) of A.integrifolia in
chronic model of epilepsy and it dose dependently prevented the occurrence of consecutive
stage 4 and/ or stage 5 seizure though the lowest dose failed to do so. During the initial
phases of the kindling process the lowest dose prevented the animals from developing
maximum stages of seizure which only lasted for a short time. This might be because of
repeated administration of PTZ caused its accumulation in the brain resulting in prolonged
antagonism of GABA making the lowest dose ineffective to restore the activity of
GABA(Corda et al., 1990).
Though the exact mechanism of the anti-convulsant activity of the study plant remains to be
elucidated, it can be generalized that the active solvent extracts can act through multiple
mechanisms because of their activity against PTZ and MES models. In fact, some of the
36
conventional anti-epileptic drugs like valproate, felbamate, topiramate and benzodiazepines
have been effective against both seizure models with broad spectrum anti-epileptic
activity(Rogawski and Löscher, 2004, Swinyard et al., 1986). This might indicate that the
study plant contains different phytoconstituents acting through different mechanisms of
action. Even if at this point we are uncertain to identify specific phytoconstituents
responsible for the anti-convulsant activity of the plant, the presence of plant derived
terpenoids, as reported in India and Iran(Kasture et al., 2002, Sayyah et al., 2002), might
have played a major role. Therefore, the anti-convulsant activity of the plant could emanate
from different types of terpenoids it contained(Kuria et al., 2002).
In addition to terpenoids, the presence of phenols perhaps contributed for the anti-convulsant
effect produced by the different solvent extracts. As reactive oxygen species play a major
role in the pathogenesis of epilepsy, the anti-oxidant activity of phenolic compounds as
reported by different investigators(Foti, 2007, Nugroho et al., 2012) could have played a
major role for the anti-convulsant effect exhibited by the study plant. In expansion to anti-
oxidant activity, studies also identified that, plant derived polyphenolic compounds have
anti-convulsant effect against PTZ induced seizure by increasing brain level of GABA
(Dhingra and Jangra, 2014). Different studies also indicated the role of alkaloids in the
treatment of epileptic seizure. Plant derived alkaloids have been found to possess anti-
convulsant activity by blocking Na+ channels. As a result, the presence of these alkaloids in
the leaf extracts of the plant(Ameri et al., 1997) could also be a major contributors for the
produced anti-convulsant effect. It was reported that a steroidal compound known as
Ergosterol-5,8-endoperoxide was isolated from the leaf part of A.integrifolia (Cantrell et al.,
1999) and studies revealed that steroidal compounds have anti-convulsant effect against
MES (Li et al., 2016) as well as PTZ induced seizure(Aliyu et al., 2014). Therefore, the anti-
convulsant activity demonstrated by the study plant could be because of this steroidal
compound present in the different solvent extracts.
In addition to the above phytoconstituents, flavonoids identified in the leaves of the plant
possibly contributed for the anti-convulsant activity obtained in different seizure models;
because studies showed that flavonoids possess anti-convulsant activity(Citraro et al., 2016).
The ubiquitous plant derived polyphenolic flavonoids were identified as one of compounds
37
with GABAA modulatory activity(Hanrahan et al., 2011). Therefore, flavonoids present in
the different extracts could be responsible for the anti-convulsant activity obtained by
enhancing GABAA mediated activity.
Studies indicated that chronic administration of PTZ increases production of reactive oxygen
species in different regions of the brain, which could be a mechanism for development of
epileptic seizure(Samokhina and Samokhin, 2018). Investigation of the leaf of A.integrifolia
for its anti-oxidant activity by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) revealed the
plant’s ability to scavenge free radicals by decreasing the concentration of DPPH by
50%(Nasser et al., 2010). Therefore, this anti-oxidant activity could be a potential
mechanism to protect the animals against PTZ induced kindling.
In addition to the anti-convulsant activity testing, determination of the major secondary

metabolites of the most active extract, ethyl acetate, revealed that it contained 21.928 ±1.118
mg GAE/ g, 9.045 ±0.8445 mg QE/g and10.002±0.119 mg atropine equivalent (AE) per
gram of dry weight of extract of phenols, flavonoids and alkaloids respectively. Methanolic
extract of aerial parts of the plant is reported to contain 5.94±1.98mg GAE/gram and 1.98
±0.06mg QE/ gram of dry extract of phenols and flavonoids, respectively(Kayani et al.,
2016). The total phenolic and flavonoid content determined in the current study was greater
than the one reported by previous study done in Pakistan. The difference in the total content
of these major phytoconstituents could be because of difference in the geographical factors
and type of solvent used as studies indicated that the use of non-polar solvent increases the
yield of total phenols and flavonoids(Bouterfas et al., 2016).
38
6. LIMITATION OF THE STUDY
The major limitation of the current study is lack of measurement of the plant’s solvent
extracts effect on EEG component of seizure, which is used to measure change in electrical
activity that occur in the brain during epileptic seizure. The other limitation is lack of
measurement of neurochemical changes (for example enzyme levels like superoxide
dismutase (SOD)) that could take place in the brain of the animals after administration of
plant extracts that would have been useful in indicating mechanism of action of the plant’s
anticonvulsant activity. In addition, inability to perform behavioral analysis after kindling
process because of scarcity of resources is another limitation of the study as PTZ kindling
can result in cognitive impairment, hippocampal neuronal degeneration and oxidative stress.
Finally, parameter recording during anti-convulsant activity testing which was carried out
manually could also be a limiting factor as it can be a source of bias.
39
7. CONCLUSION
The outcome of the present study provides support for the traditional use of Ajuga
integrifolia as anti-convulsant medicinal plant as the leaves of the plant’s solvent extracts
displayed anti-convulsant activity in both acute and chronic seizure models. The results of
this study suggest that semi-polar to non-polar components are responsible for anti-
convulsant activity of the plant while polar components are found to be devoid of anti-
convulsant activity.
40
8. SUGGESTION FOR FUTURE WORK
Anti-convulsant activity of the plant is promising; therefore, the following works are
recommended to be done in the future to make it clinically useful;
✓ The effect of plant extracts on EEG component of epileptic seizure needs to be

evaluated
✓ Isolation of pharmacologically active principles responsible for the displayed anti-
convulsant activity should be done
✓ Further studies should be done to elucidate the mechanism of action involved.
✓ More studies need to be done to analyze behavioral changes that can be seen after
PTZ kindling.
It is also good if another study is conducted to test anti-convulsant activity of roots of the
plant.
41
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Anticonvulsant activity of Soxhlet leaf extracts of Ajuga integrifolia

Buch.Ham ex D.Don (Lamiaceae) in mice

By: Tesfaye Desalegn (B.Pharm)

A thesis submitted to the Department of Pharmacology and Clinical

Addis Ababa University

Addis Ababa, Ethiopia

SCHOOL OF GRADUATE STUDIES

Signed by examining committee

Name signature date

Examiner (internal) _____________________ __________ ___________

Examiner (external) ______________________ ___________ __________

Adviser Prof. Ephrem Engidawork __________

Chair, Department of program coordinator

Anti-convulsant activity of the extracts was investigated in both acute (pentylenetetrazole,

Key words: Ajuga integrifolia, Anti-convulsant, Epilepsy, kindling, phytoconstituents,

ACKNOWLEDGEMENTS .................................................................................................................. III

LIST OF ABBREVIATIONS AND ACRONYMS .............................................................................. VI

LIST OF TABLES ............................................................................................................................... VII

LIST OF FIGURES ............................................................................................................................. VIII

1.1. Overview of Epilepsy .................................................................................................................. 1

1.2. Epidemiology of Epilepsy ........................................................................................................... 2

1.3. Pathophysiology of Epilepsy ....................................................................................................... 2

1.4. Management of Epilepsy ............................................................................................................. 6

1.4.1. Non-pharmacological treatment ........................................................................................... 6

1.4.2. Pharmacological treatment ................................................................................................... 7

1.4.3. Herbal treatment ................................................................................................................... 8

1.5. Animal models of epilepsy .......................................................................................................... 9

1.6. Overview of the experimental plant .......................................................................................... 12

1.7. Rationale of the study ................................................................................................................ 16

2.1. General objective ....................................................................................................................... 18

2.2. Specific objectives ..................................................................................................................... 18

3. EXPERIMENTAL METHODS ....................................................................................................... 19

3.1. Drugs and chemicals.................................................................................................................. 19

3.2. Experimental Animals ............................................................................................................... 19

3.3. Collection of plant material ....................................................................................................... 19

3.4. Preparation of the extracts ......................................................................................................... 20

3.5. Grouping and dosing of animals................................................................................................ 20

3.6.1. PTZ induced seizure ........................................................................................................... 21

3.6.2. MES induced seizure .......................................................................................................... 22

3.6.3. PTZ induced Kindling ........................................................................................................ 22

3.7. Quantification of phytochemical constituents ........................................................................... 23

3.7.1. Quantification of total flavonoids content .......................................................................... 23

3.7.2. Quantification of total phenolic content ............................................................................. 23

3.6.3. Quantification of total alkaloids ......................................................................................... 24

3.8. Data Analysis ............................................................................................................................ 24

4.1. Anti-convulsant activity in PTZ induced seizure ...................................................................... 25

4.2. Anti-convulsant activity in MES induced seizure ..................................................................... 27

4.3. Anti-convulsant activity in PTZ kindling Model ...................................................................... 29

4.4. Quantification of Secondary Metabolites .................................................................................. 30

4.4.1. Determination of Total flavonoids content ......................................................................... 30

4.4.2. Determination of total phenolic content ............................................................................. 31

4.4. 3. Quantification of Total alkaloid content ............................................................................ 32

6. LIMITATION OF THE STUDY ..................................................................................................... 39

8. SUGGESTION FOR FUTURE WORK .......................................................................................... 41

AED Anti-epileptic Drugs

1.1. Overview of Epilepsy

Epilepsy is divided into 4 categories based on etiology: idiopathic, symptomatic, provoked,

1.2. Epidemiology of Epilepsy

Epilepsy is also a common neurological disorder in Ethiopia with estimated prevalence of 5–

1.3. Pathophysiology of Epilepsy

1.3.2. Voltage gated ion channels

1.3.3. Changes in neuronal networks

1.3.4. Inflammatory Mediators

Investigations using experimental models identified that inflammatory processes have a

Examiner (internal) _________ _

Examiner (external) ________________ _ ______

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